Rucola plants with cytoplasmic male sterility (CMS)

ABSTRACT

The present invention discloses rucola plants, including an  E. sativa  plant, with cytoplasmic inherited male sterility (CMS) for hybrid breeding purposes. The present invention includes plants that comprise CMS-cytoplasm from cauliflower ( B. oleracea  var.  botrytis ) transferred to  E. sativa  by a wide interspecific cross.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application No. 60/845,346filed Sep. 18, 2006.

BACKGROUND OF THE INVENTION

Rucola is a traditional salad vegetable originating from theMediterranean area, which has in recent years also become very popularwith consumers in the USA and the EU. Rucola is grown and sold as leavesand is also called rocket, roquette, arugula and others. Rucola includesEruca sativa and a number of species of the genus Diplotaxis. E. sativa(“cultivated rucola”) is faster growing and more cold tolerant thanDiplotaxis species (“wild rucola”) and its taste is milder and thereforepreferable in some market segments, whereas Diplotaxis species withtheir stronger taste and longer shelf life are preferable in othermarket segments. E. sativa and Diplotaxis are both a member of theCruciferae family.

Plant breeders have worked to improve the genetic composition of E.sativa plants with a view to creating varieties that are superior toknown varieties. Examples of desired improvements are plants havingincreased yields, disease resistance, shelf life, taste, stresstolerance or earliness.

One approach to achieve this goal in vegetable crops has involvedcrossing desirable genetic traits into plants and then developing theminto pure breeding lines by successive generations of self pollination.Superior lines are then combined to form a uniform F1 hybrid thatcontains the desirable genetic traits and eliminates inbreedingdepression. In the vegetable seed markets all major vegetable crops,except lettuce, are today dominated by such superior F1 hybrids due totheir clear advantages over non-hybrids.

There is however no cost effective hybridization system available forrucola. This greatly restricts the potential to improve rucola, inparticular E. sativa plants. There is therefore an unmet need fordevelopments allowing the routine and cost-effective production ofhybrids in rucola, in particular in E. sativa.

SUMMARY OF THE INVENTION

The instant invention addresses the unmet need for developments allowingthe routine and cost-effective production of hybrid plants in rucola, inparticular in E. sativa. To meet this need the present applicationdiscloses male sterile rucola plants, in particular cytoplasmic malesterile (CMS) plants of E. sativa. The present application alsodiscloses seeds and part of these plants, and methods of producing suchplants.

Rucola plants and in particular E. sativa plants characterised by CMShave never been identified and hence it has not been possible to exploitthe opportunities given by male sterility in breeding programs in E.sativa. However, the present invention provides male sterile E. sativaplants produced by transferring a male sterility trait from anotherspecies into E. sativa through a wide interspecific cross and havesurprisingly obtained E. sativa plants with commercially acceptablegrowth and morphological characteristics. In one aspect, the flowers ofa plant of the invention have normal E. sativa morphology. In anotheraspect, seed set of a plant of the invention is in the normal range(where the number of seeds/silique is greater than 5, 8, 10, 12, 14, 16,17, 18, 19 or 20; or between 15-35 on average). In another aspect, theplant morphology of a plant of the invention is identical to E. sativa(FIGS. 8 and 9)

The introgression of the CMS trait from another species enables hybridbreeding in this crop and facilitates therefore efficient cropimprovement in order to meet emerging market needs such as enhancedyield and disease resistance, and longer shelf life. This benefits allbreeders, producers, shippers, traders and consumers of rucola products.

Accordingly the present invention, in one embodiment, provides a malesterile Rucola plant, including a plant that is Eruca sativa.

In a further embodiment said plant comprises a male sterile cytoplasmconferring male sterility upon said plant. Accordingly said plant maycomprise male sterile cytoplasm from Brassica oleracea or Brassicanapus. For example the male sterile cytoplasm may be transferred from amale sterile cytoplasm donor selected from the group consisting ofcauliflower (var. botrytis), Brussel sprouts (var. gemmifera), whitecabbage (var. capitata), oxheart cabbage (var. capitata), red cabbage(var. capitata), savoy cabbage (var. sabauda), turnip cabbage (convar.acephala DC var. sabellica), portugese cabbage (var. tronchuda), curlykale cabbage (var. sabellica), kohlrabi (var. gongylodes), broccoli(var. italica), chinese kale (var. albiflora), burma sarson (var.chinensis), kitchen kale (var. fimbriata), thousand-head kale (var.fruticosa), collards (var. sabellica) and rape seed/canola (B. napus).

In a further embodiment said male sterile cytoplasm is an “Ogura” malesterile cytoplasm and the male sterile Eruca sativa plant may comprisean orf138 DNA marker. A suitable source of said male sterile cytoplasmis from cauliflower F1 hybrid “Cheddar”.

In one embodiment the male sterile Eruca sativa plant is RQ5000/06[NCIMB no. 41429] or progeny or ancestor of said line RQ5000/06comprising said male sterile cytoplasm.

The Eruca sativa plant of the invention may be an inbred or a hybrid.

The invention includes any part of the plant including fruit, seed,pollen, ovule, embryo, leaf, stem, root or any combination thereof.

The invention further provides for use of an “Ogura” male sterilecytoplasm donor to produce a male sterile Eruca sativa plant, includingfor example a donor that is cauliflower F1 hybrid “Cheddar”.

The invention further provides a method of producing a male sterileEruca sativa plant comprising the steps of:

-   -   a) crossing a cytoplasmic male sterile Brassica oleracea or a        cytoplasmic male sterile Brassica napus plant with an Eruca        sativa plant,    -   b) rescuing one or more embryos resulting from the cross of step        a),    -   c) regenerating a plant from one or more embryos of step b) and        selecting one or more plant having Eruca sativa phenotype,    -   d) doubling the chromosome number of the plant of step c), and    -   e) back-crossing one or more plant resulting from step d) with        an Eruca sativa plant and selecting for one or plant with Eruca        sativa phenotype.

In one embodiment this method further includes testing for an orf138 DNAmarker during selecting one or plant, wherein said selected plantcomprises said marker.

A further aspect, the invention provides a method of producing a malesterile Eruca sativa plant comprising the steps of:

-   -   a. fusing a protoplast from a plant selected from cytoplasmic        male sterile Brassica oleracea, cytoplasmic male sterile        Brassice napus and cytoplasmic male sterile Eruca sativa plant,        with a protoplast from an Eruca sativa plant to produce an        allogenic cell,    -   b. regenerating the obtained allogenic cell into a cytoplasmic        male sterile E. sativa plant having cytoplasmic male sterile        cytoplasm    -   c. pollinating the regenerated plant with pollen from an Eruca        sativa plant and selecting one or more cytoplasmic male sterile        plant progeny.

In one embodiment, this method includes testing for an orf138 DNA markerwhen selecting the progeny, whereby said selected one or more plantprogeny comprises said marker.

A CMS Eruca sativa plant obtainable by any of the above disclosed methodis also encompassed by the present invention.

In a further embodiment the invention provides a method for transferringmale sterile cytoplasm to a male fertile Eruca sativa plant comprisingthe steps of:

-   -   a) crossing a cytoplasmic male sterile Eruca sativa plant with a        male fertile Eruca sativa plant,    -   b) harvesting one or more seed produced by the cross of step a),    -   c) back-crossing a plant grown from one or more seed of step b)        with said male sterile Eruca sativa plant and harvesting seed        produced by the cross,    -   d) back-crossing a plant grown from one or more harvested seed        of step c) or seed from subsequent back-crossing step with said        male sterile Eruca sativa plant for one or more back-cross        generation until one or more nuclear genes from the male        fertile E. sativa are transferred to one or more male sterile        backcross plant progeny.

According to one embodiment of this method the cytoplasmic male sterileEruca sativa plant is RQ5000/06 [NCIMB no. 41429] or progeny thereof.

The invention further provides a method of producing a leaf of a rucolaplant comprising:

-   -   a) growing a CMS rucola plant according to the invention until a        leaf is produced; and    -   b) harvesting said leaf.

Additionally the invention provides a method of vegetatively propagatinga rucola plant comprising:

-   -   a) collecting a tissue of a CMS rucola plant of the invention;    -   b) cultivating said tissue to obtain proliferated shoots;    -   c) rooting said proliferated shoots to obtain rooted plantlets.

Additionally the invention provides a method of producing seed of arucola plant comprising:

-   -   a) growing a first CMs rucola plant according to the invention;    -   b) pollinating said rucola plant with pollen of a second rucola        plant; and    -   c) harvesting seeds from said first rucola plant.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Comparison of flower morphology. Left: E. sativa cv. “Myway”;Center-left: RQ1400/04-1 (CMS cauliflower B. oleracea var. botrytis×E.sativa) F1; Center right: RQ1400/04-1, after chromosomedoubling=allotetraploid; Right: CMS cauliflower B. oleracea var.botrytis. Flowers of RQ1400/04-1 and its allotetraploid derivative(center left+right) have disfunctional anthers, which fail to produceviable pollen.

FIG. 2. Comparison of plant morphology. Left: BC₁ plant (E. sativa×B.oleracea)×E. sativa (No. RQ1438/05-1). Right: E. sativa cv. “Myway”. Itappears that the BC₁ has E. sativa serrated leaf shape but the plant ismore vigorous and leaves are larger than normal E. sativa.

FIG. 3. Comparison of leaf shapes. Left: leaf of B. oleracea var.botrytis cv. “Cheddar” Centre left: Leaf from BC₁ plant RQ 1438/05-1;Centre right: leaf of BC₂ plant RQ 5000/06. Right: Leaf of E. sativa cv.“Myway”. BC₁ and BC₂ leaves exhibit a shape similar to rucola.

FIGS. 4A-4F. DNA content of plant material measured by flow cytometry.The detectable DNA content of plant material from each of (A) E. sativacv. “Myway”; (B) Cauliflower cv. “Cheddar” and (C) R01400/04-1corresponds to a value of about 50 FL units, compared to a value ofabout 75 FL units in plant material from RQ1438/05-1 (D), indicatingthat RQ1438/05-1 has 3 genomes, i.e. is triploid. (E) BC₂ plant RQ5000/06-3 is diploid, as also observed in plants RQ 5000/06-1, -2 and-4, whereas plant RQ 5000/06-5 (F) appears to have 3 chromosomal setsfrom E. sativa and 1 from B. oleracea. X-axis (FL1)=fluorescenceintensity units and count=total counts.

FIGS. 5A-5F. CMS marker detection in CMS-E. sativa and parent lines. PCRproducts from amplification of plant DNA extracts using primercombination oligo 37+oligo 38, are analysed on ethidium bromide-stainedagarose gels. The 512 bp fragment indicates the presence of “Ogura” CMSand the 630 bp fragment is a positive control for the presence of thecob gene and a positive control for the PCR reaction. PCR products fromDNA extracted from plant samples are shown as follows: A: Lanes 1, 4band 5 are from samples of “Ogura” CMS cauliflowers; lanes 2 and 3b arecauliflowers lacking “Ogura” CMS; lanes 6, 7 and 8b are normalmale-fertile E. sativa cultivars; lanes 9-16 are different allodiploidCMS-E. sativa plants, and lane 4b is cv. “Cheddar” the CMS donor used togenerate the CMS-E. sativa plants in lanes 9-16. Lane M comprises DNAsize markers.

FIG. 6: Normal fertile and male sterile “Ogura” flower structure. Right:normal E. sativa anthers with pollen. Left: Anthers from CMS+E. sativaplant without pollen.

FIG. 7: Magnified image of normal fertile and male sterile “Ogura”anther structure. Left: normal fertile E. sativa anther with pollen.Right: CMS-E. sativa anther.

FIG. 8: Left: Normal flower of E. Sativa. Right: CMS-E. sativa flower(Line 5000/06). Anthers are shrunken and thin, i.e. not carrying pollen.

FIG. 9. Comparison of plant morphology. Left: CMS-E. sativa BC₃ plant.Right: E. sativa cv. “Myway”. It is seen that the plants are almostidentical.

FIG. 10. Nucleotide sequence of “orf138”. Nucleotide sequence (2427 nt)of “orf138” gene corresponding to Brassica sp. cybrid mitochondrial DNAORF158, ORF138 and tRNA-fMet: ACCESSION Z12626. PCR detection of“orf138” DNA marker employs an upper primer (Oligo 37) and lower primer(oligo 38) which amplify a DNA sequence corresponding to nucleotides 937to 1449 of the shown sequence (highlighted in grey). The annealingposition of the primers in the nucleotide sequence is indicated byunderlining. PCR amplification with oligo 37 and 38 yields a 512 bp DNAproduct.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Agglutination: The formation of clumps of cells.

Allogenic: Genetically different.

Alloplasmic: Organism containing cytoplasm (including mitochondria andchloroplasts) from one species and nucleus from another.

Allotetraploid: Tetraploid produced from a hybrid between two or moredifferent species and therefore possessing two or more different sets ofchromosomes.

Amphidiploid: Diploid produced from a hybrid between two differentspecies and therefore possessing two different sets of chromosomes.

BC: Back Cross. Classical breeding technique that can be used tointroduce the cytoplasm (or genes) from a donor into a homologousgenetic background called the recurrent parent (RP). The female donor iscrossed once with the RP, hereby a F1 hybrid is created. The F1 hybridis then crossed with the RP giving the BC₁ generation. BC₁ is crossedwith RP etc. This is repeated several times. Thereby the homologousgenetic line is recreated within the cytoplasm from the donor.

Callus: Mass of undifferentiated cells that initially arises from plantcell or tissue in artificial culture.

CMS: A form of male sterility in plants determined by cytoplasmicfactors, usually mitochondrial DNA.

CMS cytoplasm: Cytoplasm containing male sterility determining factors.

Cytoplasm: All the living part of a cell inside the cell membrane andexcluding the nucleus.

Cytoplasmic locus “orf138”: A specific marker (from INRA) for the“Ogura” male sterility. The primer combination amplifying this markerhave the sequences:

Oligo37: GCA TCA CTC TCC CTG TCG TTA TCGOligo38: ATT ATT TTC TCG GTC CAT TTT CCA

The expected amplification product is 512 bp. The sequence of the orf138gene is available on the public databases (NCBI) at accessionN^(o)Z12626.

Endonuclease: Nuclease enzyme that splits DNA at internal sites. Byusing such enzymes on DNA, a certain unique digestion pattern isobtained. This pattern can be used to distinguish genetically differentorganisms.

Eruca sativa phenotype: is a phenotype characterised by a range offeatures that distinguish the morphological growth habit of Erucastativa plants, including leaf shape and size, frequency of stembranching, plant height and volume, flower shape and size as illustratedin the figures. Selection of a plant with Eruca sativa phenotype canthus include the selection of a plant comprising one or more of thesedistinguishing morphological features.

F1 hybrid: Seeds or plants originating from a cross between two inbredparent lines. F1 denotes 1^(st) filial generation.

Feeder cells: Cells used in e.g. protoplast culture in order to supportthe development of the fragile protoplasts.

Gamete: Haploid reproductive cell produced by sexually reproducingorganisms, which fuses with another gamete of opposite sex or matingtype to produce a zygote.

Flow cytometry: Technique for counting cells and distinguishingdifferent types of cells in a mixed population. The cells are usuallystained with different flourecent antibodies to distinctive cell surfacemolecules and a stream of labeled cells is then run through afluorescence detector, which counts the cells of each type. The methodcan also be used for determining the amount of DNA in the cells andthereby give an indication of the ploidy level.

Heterokaryon: A cell containing two (or more) genetically differentnucei, formed naturally or artificially in culture by the fusion of twoanimal cells or plant protoplasts.

Inbred: line produced by successive self pollinations.

Interspecific hybrids: Hybrid made from a cross between two differentspecies.

Introgressing: Gradual transfer of a gene from one line to another e.g.by back crossing.

Maintainer plant line: Male fertile plant line used as pollinator forseed multiplication of a specific CMS line. The cytoplasm of themaintainer line is male fertile, but the nuclear chromosomes areidentical to the nuclear chromosomes of the specific CMS line.

Male fertile: Organism or plant able to produce viable male gametes.

Male sterile: Organism or plant unable to produce viable male gametes.Partly male sterile means that the amount of viable pollen isinsignificant with respect to potential for commercial hybrid seedproduction from a given female line.

Meiotic pairing: Stage in the meiosis (the process producing gametes),where the homologous chromosomes are aligned on the equator of the cellwith all the centromers lying along the spindle equator.

MES: 2-(N-Morpholino) ethanesulfonic acid.

Micro calli: Very small pieces of callus.

Micro-manipulation: Common technique used to handle and sort singlecells.

Microsatellite marker: See SSR marker

Mitochondria: Organelles in the cytoplasm of eukaryotic cells, having adouble membrane, the inner invaginated, and which are the site of thetricarboxylic cycle and oxidative phosphorylation of oxidativerespiration, generating ATP. They contain a small circular DNA whichspecifies tRNA's, rRNA's and some mitochondrial proteins.

n: Haploid chromosome number.

Nucleus: A large dense organelle bounded by a double membrane, presentin eukaryotic cells, and which contains the chromatin and in which theDNA replication and transcription takes place.

NCIMB: National Collection of Industrial Bacteria. The collection alsoaccepts seeds.

Ogura CMS: The term Ogura CMS cytoplasm as used herein refers toRaphanus sativus originating cytoplasm comprising mitochondrial DNAwhich confers male sterility to plants. The term Ogura CMS Brassicaoleracea plant or plant cell as used herein refers to a Brassicaoleracea plant or plant cell comprising Ogura CMS cytoplasm.

Osmolarity: The osmotic concentration of a solution.

Osmoticum: Compound used to increase the osmotic concentration of asolution

Ovule: In seed plants, the structure consisting of the megagametophyteand megaspore, surrounded by the nucellus and enclosed in an integument,and which develops into a seed after fertilization.

PCR: Polymerase Chain Reaction. Common technique used to amplifyspecific regions or fragments of DNA.

Primer: Short specific DNA fragment, in the PCR, a pair of syntheticoligonucleotides (primers) complementary to flanking regions of the DNAto be copied, which are bound to the DNA before the reaction commencesto ensure that DNA replication is initiated at the required points.

Ploidy-level: Number of chromosomes or DNA molecules in a cell ororganelle, or the typical chromosome number of a multicellular organism.

Protoplast: plant cell with cell wall removed; the living component of acell, i.e. the protoplasm not including any cell wall.

SSR marker: Simple Sequence Repeat. Certain areas of the genomecontaining repeated simple sequences e.g. ATT n times. These repeats areknown to have a high degree of polymorphism and are therefore useful asDNA markers.

Triploid: An organism with three sets of chromosomes per somatic cell.

Exploiting hybrid opportunities in rucola would significantly increasethe efficiency of rucola breeding, since in-breeding depression of anumber of traits could then be eliminated.

One method of producing hybrids relies on male sterility in the breedingline for which hybridization is desired. Male sterile lines allow thebreeder to produce hybrid seed by controlling cross-fertilization in theflowers of the breeding line (i.e. mother line). Cross-fertilization issecured and self-fertilization eliminated by using breeding line plantsthat are male sterile due to their failure to produce viable pollen.100% hybridization of the male sterile breeding line with the desiredfather line can then be obtained.

Several sources of CMS can be used in the context of the presentinvention, for example: “Ogura” male sterile cytoplasm of Raphanussativus); “Polima” male sterile cytoplasm of B. napus, and “Nap” malesterile cytoplasm of B. napus and “Anand” male sterile cytoplasm ofBrassica toumefortii (Cardi, T. & E. D. Earle, 1997).

Plants carrying CMS can become male fertile again, or partly malefertile, in the presence of certain nuclear genes. These nuclear genesare called “restorer genes” because they counteract the effect of theCMS cytoplasm. Male fertility can be restored in the F1 hybrid bycrossing with a pollinator line carrying restorer genes.

Crosses between different species of Cruciferae would facilitate thetransfer of desirable genes, such as cytoplasmic male sterility, betweenspecies. However, the production of interspecific hybrids is generallyunpredictable and often extremely difficult to accomplish due to theneed to overcome the natural genetic barriers that exist betweenspecies.

Another way of producing interspecific hybrids, comprising cytoplasmicgenes from one species and nuclear genes from another, is by protoplastfusion. Here a protoplast from a species having desirable traits iscombined with a protoplast from a CMS plant from another species. Thenuclear material of the CMS donor is removed, or inactivated, prior tofusion, so it donates only the cytoplasm. The resulting cytoplasmichybrid or “cybrid” is then re-generated into a plant. This plant willpossess the nuclear genes for the desirable traits and be male sterile.This procedure is as unpredictable for the generation of interspecifichybrids as the crossing method outlined above.

The barrier for obtaining a true interspecific hybrid can either becaused by the inability of the foreign pollen tubes to penetrate intothe ovarian tissue of the pollen acceptor, or be caused by eliminationof all the chromosomes of one of the parents, typically the father. In across between E. sativa and B. oleracea, Yadav et al. 2002 showed thatE. sativa did not function as a pollen acceptor. In the reciprocalcross, limited fertilization occurred, and only few pollen tubes wereseen in the ovarian tissue of B. oleracea after 24 h of pollination.Sundberg & Glimelius, 1991 showed that in a cross between B. napus andE. sativa, the chromosomes of the E. sativa genome appeared to bepreferentially sorted out. A similar mechanism was observed in theinitial B. oleracea×E. sativa crosses made for this invention, in whichonly a few plants, derived from embryo-rescued seeds, phenotypicallyresembled true interspecific hybrids between E. sativa and B. oleracea.The rest of the plants were phenotypically similar the B. oleraceafemale line. The production of CMS rucola plants (e.g. CMS E. sativa)according to the present invention has thus surprisingly been achievedas detailed below.

Seeds of line RQ5000/06, a representative plant according to the presentinvention, have been deposited with NCIMB ltd., Bucksburn, Aberdeen,AB21 9YA, on Aug. 14, 2006 under accession number NCIMB 41429.

I. Crossing Program for Production of CMS E. sativa

The present invention discloses rucola plants (e.g. E. sativa plants)with CMS, introgressed from B. oleracea. E. sativa, itself, cannot beused as a CMS donor to obtain a CMS rucola hybrid, because the CMS traithas never been reported in these plants.

Interspecific hybridization is performed by crossing a CMS donor asmother such as cauliflower (var. botrytis), Brussel sprouts (var.gemmifera), white cabbage (var. capitata), oxheart cabbage (var.capitata), red cabbage (var. capitata), savoy cabbage (var. sabauda),turnip cabbage (convar. acephala DC var. sabellica), portugese cabbage(var. tronchuda), curly kale cabbage (var. sabellica), kohlrabi (var.gongylodes), broccoli (var. italica), chinese kale (var. albiflora),burma sarson (var. chinensis), kitchen kale (var. fimbriata),thousand-head kale (var. fruticosa), collards (var. sabellica) and fromrape seed/canola (B. napus), and E. sativa as father. When CMS isintrogressed from a cauliflower (B. oleracea var. botrytis) donor, itprovides favourable flower morphology. Since cauliflower (n=9) has adiploid genome of 18 chromosomes, and E. sativa (n=11) has a diploidgenome of 22 chromosomes, they are not sexually compatible. In thedeveloping seed this incompatibility can result in incompletedegradation of the endosperm needed to support embryo maturation, andthe loss of chromosomes from the pollinator. The growth and survival ofembryos produced by the cross may however be secured by employing the“embryo rescue” technique.

Embryo rescue involves removing the embryo from the silique produced bythe cross, about 3 weeks after pollination, the exact timing of embryorescue depending on the time of the year and the parent lines in thecross. The siliques are first treated with a disinfectant and thenrinsed in sterile water to eliminate microbial contamination. Thesiliques are then cut open and the embryos are removed and placed on asuitable medium to facilitate growth, as for example MS (Murashige &Skoog medium) detailed in Table 1. The embryos are then grown atapproximately 25° C. in a dark/light regime until they have germinatedand developed roots, after which they are transferred to soil and grownunder a day/night regime of about 20° C. night and 22-25° C. day.

Regenerated F1 plants are grown to maturity, and those producing flowerswith dysfunctional anthers lacking pollen are expected to carry the CMStrait. F1 plants that have both the CMS trait and exhibit desired traitsconferred by the paternal parent may be identified by their phenotypicproperties. Growth properties and morphology that may be selected forinclude vigorous growth, shelf-life (increased lead thickness andsurface wax), flavour (bitterness), disease resistance, dry mattercontent, low nitrate content). By way of example, a cross between CMS B.oleracea and E. sativa produced an F1 plant (plant no. RQ1400/04-1),whose growth was more vigorous than a normal rucola, but its volume wassmaller than a cauliflower. The leaves were serrated like the parentaltype of E. sativa during the entire growth. The flowers of the plantclearly carried the CMS trait, producing disfunctional anthers withoutpollen, but otherwise having a phenotypically normal cruciferous flowertype (FIG. 1, second from left) when compared to flowers of the twoparental lines of the cross (FIG. 1, left and right). Despite conductinga very large number of crosses between the RQ1400/04-1 plant and E.sativa pollinators, throughout the entire flowering period, no seeds oreven embryos were obtained.

The chromosome complement of the hybrid F1 plants generated from a crossbetween a B. oleracea CMS donor and an E. sativa cv can be determined byextracting nuclei from the hybrid plant material and measuring theamount of DNA by flow cytometry. Different protocols for the isolationof nuclei and preparation of stained nuclei suspensions for flowcytometry can be used, as exemplified in example 1. As shown in Example1, the DNA content of RQ1400/04-1 was consistent with an amphihaploidplant, having one set of 9 chromosomes from cauliflower and one of 11from E. sativa (FIG. 4). Such plants can be propagated vegetatively, andthe clones maintained in vivo, however they may be found infertile whencrossed to other E. sativa pollinators. If the chromosomal complement ofa hybrid F1 plant comprises a cauliflower chromosome set and an E.sativa chromosome set, as is the case for RQ1400/04-1, then the plant islikely to be infertile due to an inability to conduct normal meioticpairing and form fertile gametes as required for subsequent formation ofprogeny. In this case it is desirable to double the chromosomecomplement of the hybrid F1 plant to generate a plant with 2 sets ofcauliflower chromosomes and 2 sets of E. sativa chromosomes. Suchchromosome-doubled plants are capable of undergoing meiosis since the 2sets of chromosomes can perform chromosomal pairing internally and theplants are thus fertile. A chromosome doubling strategy, employingcolchicine treatment, was successfully employed to generate a fertilederivative of vegetative clones of RQ1400104-1 plants (Example 1D).

Seedlings of CMS hybrid F1 plants that have undergone successfulchromosome doubling are recognised by their distinctive phenotype,including vigorous growth of stems and leaves, and flowers of up todouble their normal size, while retaining their CMS-phenotype withrudimentary anthers devoid of pollen. During the flowering period, thesecolchicine-treated plants may be back-crossed with E. sativa cultivarsto produce viable seed. Progeny plants are selected for the CMS traitand for inheritance of desired E. sativa traits including for exampleserrated leaf habit, E. sativa flower morphology, vigorous growth,fertility, high seed set, shelf-life (increased lead thickness andsurface wax), flavour (bitterness), disease resistance, dry mattercontent, low nitrate content).

The chromosome complement of a selected hybrid back-cross may bedetermined by extracting nuclei from a sample of hybrid plant materialand flow cytometry. The selected back-crossed progeny (BC₁) may, forexample, be found to be triploid with a chromosome complement consistentwith one set of cauliflower chromosomes (n=9) and 2 sets of E. sativachromosomes (n=11) giving a total of 31 chromosomes as seen for BC1lines derived from RQ1400/04-1 plants (Example 1D).

DNA marker analysis, may be employed to demonstrate the presence of the“Ogura” CMS in the BC_(>1) progeny, as illustrated using the “orf138”marker in example 13+FIG. 5.

The selected BC₁ generation may have a reduced fertility and may nothave all the desired traits from the E. sativa parent, in which case aback-crossing program may be performed between the selected BC₁ plantand the desired E. sativa paternal line. As illustrated in Example 1,the back crossing of the triploid BC₁ plant RQ1438/05-1 to E. sativa cv.“Myway” generated a number of BC₂ plants, whose morphology closelyresembled E. sativa as well as retaining male sterility. Flow cytometryanalysis indicated that these BC₂ plants had reverted to diploidchromosome complement.

Hence a back-crossing program starting from the selected BC₁ plants cangenerate plant lines that yield similar number of seeds as theirrecurrent parent E. sativa and produce leaves compatible with commercialleaf production, as illustrated for the BC₂ plant RQ1438/05-1 in Example1 and FIG. 9.

The amount of B. oleracea DNA still present in the BC_(>), plantsgenerated from back-crossing, may be determined by DNA marker analysis,for example by using 72 SSR markers covering the whole C-genome of B.napus was performed. In Example 14, the CMS BC₃ plants were shown tohave a very high homology to E. sativa, and that all of the B. oleraceachromosomes had been eliminated in the back-crossing process.

II Protoplast Fusion Program

CMS E. sativa plants can also be prepared by protoplast fusion. Theprotoplasts can be obtained from a B. oleracea or B. napus plant thatcontains the “Ogura” CMS cytoplasm and is male sterile and E. sativaplants with desirable agronomic traits. Suitable CMS donor plantsinclude B. oleracea plants e.g. cauliflower cv. “Cheddar” or other CMSB. oleracea or B. napus varieties.

Protoplasts can be obtained from green plant material e.g. leaf materialand from white plant material e.g. etiolated seedlings, according to themethod described in example 2 (Glimelius (1984), Physiologia Plantarum61:38). When protoplasts are isolated from white plant material it isadvantageous to stain it with a flourescent dye to facilitate selectionof the best material, see example 2C. After protoplast isolation thenucleus in the CMS containing protoplast must be inactivated byirradiation.

The inactivation of the nucleus by irradiation can be effected with theaid of gamma, UV or X-rays. Where irradiation is effected with an X-raysource, nucleus inactivation will in general be obtained by applying adose of e.g. 10 krad. min for 3 to 20 minutes. The appropriate X-raydosage may for example be established by determining the maximum levelof X-ray irradiation killing 100% of the protoplast population: thepercentage of dead cells is estimated by counting the number of formedcolonies after 10 to 20 days in culture. Based on the optimum levelX-ray irradiation required, protoplast fusion is performed with anirradiated CMS protoplast (with inactivated nucleus) and the E. sativaprotoplast.

Fusion of isolated protoplasts may then be accomplished by employingpolyethylene glycol (PEG) which causes agglutination, in the presence ofa high pH fusion buffer, which promotes membrane fusion betweenprotoplasts. Somatic hybridization may be performed under the conditionsdisclosed by Sundberg et al., (Plant Science, 1986, 43:155), herebyincorporated by reference, for the production of interspecific hybridsor modifications thereof. However, one skilled in the art wouldrecognize that protoplast fusion can be accomplished by means other thanusing polyethylene glycol (PEG). For example, the protoplasts can befused by using electric field-induced fusion techniques as described byKoop et al. in Electroporation and Electrofusion in Cell Biology,edited: Neuman et al. pgs 355-365 (1989), herewith incorporated byreference. Additionally, protoplast fusion can be accomplished withdextran and polyvinyl alcohol as described by Hauptmann et al.,“Carrot×Tobacco Somatic Cell Hybrids Selected by Amino Acid AnalogResistance Complementation”, 6th International Protoplast Symposium,Basel, Aug. 12-16, 1983, herewith incorporated by reference.

If protoplast fusion is to be accomplished with polyethylene glycol, theprocedure described below can be used.

The protoplast fusion is conveniently effected in a washing solution(W5′), described below, containing an osmoticum e.g. a carbohydrate suchas mannitol, sorbitol, glucose, or sucrose, and potassium and calciumsalts. The pH can range from 5.2 to 10 and is preferably about 5.7. Theprotoplasts of different origin are mixed and concentrated, convenientlyto a final density of 1×10⁵ and 1×10⁸ protoplasts per ml.

The protoplast mixture should then be left undisturbed in a container(e.g. petri dish) for at least 10 minutes to allow the protoplasts tosettle at the bottom of the container. The mixture is then treated withpolyethylene glycol (PEG), preferably having a molecular weight of 1500to 6000. In general, good results are obtained when employing an aqueoussolution (PFS) comprising 18.8% by weight of PEG at a volume ratio ofW5′ to PFS of 10:1 to 1:1. PFS comprises conveniently an osmoticum and acalcium salt. The protoplasts are incubated in PFS for 15 to 20 minutesdepending on the fragility of the cells.

The fusion is accomplished by washing the protoplasts, for example,three times, with washing solution (W5′) containing an osmoticum (e.g.glucose) in a concentration having a lower osmolarity than PFS andpotassium, sodium and calcium salts. The fusion procedure is carried outat a temperature in the range between 16° C. and 20° C., for example 18°C. The concentration of PEG in the fusion mixture is gradually decreasedwith each consecutive washing step (see e.g. example 10). Each washingstep should take at least 5 minutes to allow the protoplasts to adjustslowly to the lower osmolarity of the medium, and avoid the protoplastsbursting. After the washing steps have been accomplished, the fusedprotoplasts should be in the range of 1×10⁵ to 1×10⁶ protoplasts per ml.

Hybrids can then be regenerated in the presence of non-fused parentalprotoplasts or after their isolation from the culture by selection.

Selection of the hybrids from the non-fused protoplasts can be done bystaining and seperation by a micromanipulator or by flow cytometrycontaining a cell sorting function.

If the protoplasts are stained with fluorescein diacetate the protoplastof hypocotyl origin will stain yellow under a UV light and protoplastfrom leaves containing chloroplast will give a red auto-fluorescensunder UV light (Sunderberg and Glemelius. 1986, Plant Science43:155-162; Glimelius et al., 1986, Plant science 45:133-141).

The obtained fusion products can be cultivated in an appropriate culturemedium comprising a well-balanced nutrient supply for protoplast growth.The medium contains micro- and macro-elements, vitamins, amino acids andsmall amounts of carbohydrates, e.g. various sugars such as glucose.Glucose serves as a carbon source as well as an osmoticum. The culturemedium also comprises plant hormones (auxins and cytokine) which areable to regulate cell division and shoot regeneration. Examples ofsuitable auxins include naphtyl acetic acid (NAA),2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA).Examples of suitable cytokinins include benzyl aminopyrine (BAP), zeatin(Zea) and gibberellic acid (GA3). In general NAA and 2,4-D are used incombination with BAP to initiate cell division, in which case the ratioof auxin/cytokinin must be high, for example greater than 1. Two orthree days after the fusion treatment, the medium is largely replaced byculture medium (BP) which comprises agarose, in which the fusionproducts and the parental protoplasts are embedded.

After 14 days, the concentration of auxins is diluted by addition of aculture medium containing no or substantially-less auxins. Star-shapedmicro calli will generally develop after 3 to 4 weeks. Such micro calliare then transferred to a regeneration medium to initiate shootformation, preferably after adaption in an intermediate regenerationmedium to allow the cells to adjust to differences in the compositionand physical properties of the culture medium and the regenerationmedium. In order to induce shoot formation, the auxin/cytokinin ratio inthe regeneration medium should preferably be low, e.g. below 1:10. Ingeneral it will be preferable to employ the auxin NAA in combinationwith the cytokinins Zea and BAP for shoot regeneration. The nutrientcontent of the regeneration media, BR and K3, is relatively compared tothat of the culture medium, since they contain less vitamins, a lower ofcarbon source content comprising solely sucrose and xylose, and do notcontain amino acids. The regeneration media also have a higher viscositythan the culture medium. The regeneration medium Br is a solid mediumand contains the growth regulators 2,4-D, NAA and BAP, with the ratio ofauxin to cytokinin being less than 1. Medium K3 contains Zea, GA3 andalso silver nitrate to promote the shoot development.

After two weeks regeneration on Br medium, calli of approximately 3 mmin diameter are transferred to K3 regeneration medium containing a lowsucrose concentration. At this stage shoots will develop within 2 to 3weeks. The obtained shoots are then rooted on a basic medium, such asB5, without additional hormones. The nuclear DNA and mitochondrial DNAof the obtained plantlets may then be identified by standard methodsknown in the art, e.g. employing suitable restriction endonucleases andcomparing the thus obtained DNA digestion pattern of DNA from the fusionproducts with that of the parental lines.

As described above, after the fusion, the hybrid cells are regeneratedto form E. sativa plants containing the CMS cytoplasm. These plants maybe subsequently back-crossed with other E. sativa plants.

It will be appreciated that the E. sativa plant of this invention may beemployed as starting material for the preparation of other E. sativavarieties having CMS by in vitro and/or crossing techniques. Such invitro and crossing techniques are known in the art by the skilledbreeder.

EXAMPLE 1 CMS E. sativa Generated by a Crossing Program

Interspecific hybridization was carried out using an orange cauliflowerF1 hybrid “Cheddar” (Seminis Vegetable Seeds) as mother, and E. sativacv.

“Myway” (L. Dæhnfeldt A/S) as father. Numerous interspecific crosseswere performed of which a total of 10 F1 plants were regenerated byemploying the following embryo rescue procedure:

A. Embryo Rescue

Approximately 3 weeks after cross-pollination, the siliques wereharvested from the pollinated parent plant. The siliques weredisinfected for 20 minutes in a 2.5% corsoline solution and rinsed threetimes in sterile water. The siliques were then cut in a longitudinaldirection and the embryos removed and plated on MS medium (Table 1),with 3% sucrose and without hormones, in a plastic container. Theembryos were then grown at 25° C. in 16 h light for at least two weeks.When the embryos had germinated into plants and developed roots theywere subcultured on peat, covered with white plastic, and grown for 5-7days in a greenhouse under 20° C. night and 22-25° C. day regime.

TABLE 1 Composition of embryo growth and regeneration media (1 |). MSMAC 8P Br K3 B5 CaCL₂•2H₂O 440 mg 600 mg 600 mg 300 mg 300 mg 150 mgCoCl₂•6H₂O 0.025 mg 0.025 mg 0.025 mg 0.025 mg 0.025 mg 0.025 mgCuSO₄•5H₂O 0.025 mg 0.025 mg 0.025 mg 0.025 mg 0.025 mg 0.025 mg FeNaEDTA 40 mg 40 mg 40 mg 40 mg 40 mg 40 mg H₃BO₃ 6.20 mg 3 mg 3 mg 3 mg 3mg 3 mg KH₂PO₄ 170 mg 164 mg 164 mg KI 0.83 mg 0.75 mg 0.75 mg 0.75 mg0.75 mg 0.75 mg KNO₃ 1900 mg 956 mg 956 mg 1556 mg 1556 mg 3000 mgMgSO₄•7H₂O 370 mg 300 mg 300 mg 250 mg 250 mg 250 mg MnSO₄•4H₂O 22.3 mg10 mg 10 mg 10 mg 10 mg 10 mg Na₂MoO₄•2H₂O 0.025 mg 0.025 mg 0.025 mg0.025 mg 0.025 mg 0.025 mg NH₄NO₃ 1650 mg 600 mg 600 mg 250 mg 250 mgZnSO₄•7H₂O 8.6 mg 2 mg 2 mg 2 mg 2 mg 2 mg Myo-inositol 100 mg 100 mgThiamine 0.10 mg 10 mg 10 mg 10 mg 10 mg 10 mg Pyridoxine 0.5 mg 1 mg 1mg 1 mg 1 mg 1 mg Nicotinic acid 0.5 mg 1 mg 1 mg 1 mg 1 mg 1 mg Glycine2 mg Sodium 20 mg 20 mg Pyruvate Citric Acid 40 mg 40 mg Maleic acidFurnaric acid 40 mg 40 mg Glucose 40 mg 40 mg Casein 250 mg 250 mg NAA0.1 mg 0.1 mg 0.1 mg 2,4D 0.2 mg 1 mg 0.1 mg Zeatin 0.26 mg BAP 0.5 mg0.5 mg 0.5 mg GA₃ 0.006 mg AgNO₃ 5 mg Xylose 250 mg Sucrose 30 g 80 g 50g 40 g 10 g 20 g Agar 8 g 8 g 8 g Agarose 16 gB. Phenotypic Properties of F1 Hybrid Plants

From the 10 regenerated F1 plants obtained from the interspecific cross,five clearly exhibited a cauliflower phenotype whereas the other fiveclearly exhibited the serrated leaf phenotype of E. sativa. Only one ofthese latter plants survived to the sexual stage (plant no.RQ1400/04-1). During the entire growth, the phenotype of this singleplant closely resembled a cross between E. sativa and cauliflower. Itwas more vigorous than a normal rucola, but the volume of the plant wassmaller than a cauliflower. The leaves were serrated like the parentaltype of E. sativa during the entire growth. The flowers of the plantclearly carried the CMS trait, producing disfunctional anthers withoutpollen, but otherwise having a phenotypically normal cruciferous flowertype (FIG. 1, second from left). This plant was propagated vegetatively,and the clones maintained in vivo.

C. DNA Content of Hybrid Plants

In order to determine the chromosome content of hybrid lines produced bythe interspecific cross of orange cauliflower F1 hybrid “Cheddar” and E.sativa cv. “Myway”, DNA was extracted from leaf material of the plantsand analysed by flow cytometry according to the following protocol.

A small piece of the leaf sample (0.5 cm²) was chopped with a sharpscalpel in a petri dish containing 2.5 ml of a detergent buffer (table2). The suspension of nuclei released from the cut tissue was filteredthrough a 50 mm nylon mesh to remove large tissue and cellularfragments. After 5 minutes of incubation, 5 ml of a staining solution(table 3) was added to the sample and after 10 minutes of incubation thesample was ready to be analysed by using Flow cytometer (Partec GmbH,Munster, Federal Republic of Germany). The sample of stained nuclei aretransferred to sample tube, placed in the sample holder on the Flowcytometer, and the DNA content is measured and plotted in histogramformat.

The DNA content of RQ1400/04-1 was consistent with an amphihaploidplant, having one set of 9 chromosomes from cauliflower and one of 11from E. sativa (FIG. 4 C compared to the DNA content of the parentallines shown in 4 A and B).

TABLE 2 Detergent solution (100 ml) 100 ml distilled water 2.1 g citricacid•H₂O 0,5 ml Tween 20

TABLE 3 Staining solution (100 ml) 100 ml distilled water 7.1 gNa₂HPO₄•2H₂O 0.2 mg DAPI (4,6-diamidino-2-phenylindole)D. Producing a Fertile Derivative of CMS F1 Hybrid RQ1400/04-1

A very large number of crosses (>1000) between the RQ1400/04-1 plant andE. sativa pollinators were made, throughout the entire flowering period,however no seeds or even embryos were obtained. Infertility wasattributed to amphihaploid chromosome complement of the RQ1400/04-1plant, and the inability to conduct normal meiotic pairing duringgametogenesis. The plants were thus treated with colchicine, accordingto the following protocol, to produce fertile plants with a doubledchromosome complement, capable of undergoing normal meiosis.

Vegetative clones of RQ1400/04-1 plants, comprising 10 cm rootedseedlings, were first washed in water and then dipped in a 0.34% (WN)solution of colchicine for 3 hours. The seedlings were then rinsed andpotted in a sphagnum mix in a ratio of 1000 liter spaghnum (type “BrunStenrøgel”) to 1 kg of CaCO₃ (chalk) [supplied by Stenrøgel A/S,Stenrøgelvej 13, Thorning, 8620 Kjellerup, Denmark].

On reaching the flowering stage, approximately 50% of the plants had aphenotype characteristic of plants following chromosome doubling,including extraordinary vigorous growth of stems and leaves, and flowersof almost double their normal size (FIG. 1, center right). The flowersof these plants retained their CMS-phenotype with rudimentary anthersdevoid of pollen (FIGS. 6 and 7). During the flowering period, thesecolchicine-treated plants were crossed extensively (>2.000 pollinations)with the E. sativa cultivars “Runway” and “Myway”. Progeny were obtainedfrom these crosses comprising a total of only 4 seeds from the cv.“Runway” crosses and 4 seeds from the cv. “Myway” crosses. The seedswere harvested but only 1 seed from the cv. “Runway” cross (No.RQ1437/05-1) and 2 seeds from the cv. “Myway” cross (No. RQ1438/05-1 and-2) germinated. These plants were again clearly more vigorous than anormal rucola (FIG. 2). The leaves were serrated like the parental typeof E. sativa during the entire growth (FIG. 3).

The surviving back-crossed progeny (BC₁) were all found to be triploidon the basis of DNA analysis by flow cytometry (see FIG. 4D compared toA,B and C), with a chromosome complement consistent with one set ofcauliflower chromosomes (n=9) and 2 sets of E. sativa chromosomes (n=11)giving a total of 31 chromosomes.

The phenotypes of RQ1438/05-1 and -2 plants were very similar to that ofE. sativa, but their vegetative growth was more vigorous (see FIG. 2 andFIG. 3), and their conversion to the sexual stage was also very severelydelayed compared to E. sativa. RQ1438/05-1 was the first of the 3 plantsto flower, and expressed a male-sterile phenotype, while otherwisehaving normal flower morphology. DNA marker analysis revealed that all 3plants contained the “orf138” marker, showing that the “Ogura” CMS ispresent (See example 13+FIG. 5).

E. Back-Crossed Lines Derived from RQ14318/05 Plants Comprising E.sativa and CMS Traits

RQ1438/05-1, of the BC₁ generation, was pollinated extensively with cv.“Myway” and 3 month after the first pollination a total of 33 BC₂ seedswere harvested, consistent with a low fertility.

The 5 first mature BC₂ seeds (Line no. RQ 5000/06) were germinated and 5individual BC₂ plants were obtained, all of which were male sterile. Ofthese plants, number 1 to 4 clearly resembled the E. sativa morphology,whereas number 5 was more vigorous. Apart from having dysfunctionalanthers, the flowers of the BC₂ plants were now morphologically similarto E. sativa var. “Myway” (FIG. 8).

A flow cytometry analysis suggested that the plants 1, 2, 3 and 4 hadreverted to diploid chromosome complement, and that plant number 5 wasamphitetraploid carrying 3 sets of E. sativa and 1 set of B. oleraceachromosomes 3n=33+n=8=41 chromosomes. (FIGS. 4E-F). DNA marker analysisrevealed that all 5 plants of RQ 5000/06 contained the “orf138” marker,showing that the “Ogura” CMS is present (Example 13+FIG. 5).

F. Diploid CMS E. sativa Derived from Back-Crossing of RQ1438/05 Plants

The four 4 BC₂ plants, identified as diploid E. sativa, yielded anaverage of 24.9 seeds/silique, which compares favourably with the malefertile E. sativa used as RP (Recurrent Parent), that yields an averageof 17.3 seeds/silique. The amount of seed produced was normal for E.sativa in relation to the time of the year and the number ofpollinations made, and on this basis it was concluded that femalefertility was fully restored in the BC₃ plants. From these BC₃ seeds adeposit was made at NCIMB with Accession No. 41429. The BC₃ plantsgenerated from RQ 5000/06 are indistinguishable from the recurrentparent E. sativa (cv. “Myway”) at the harvest size normally used forcommercial leaf production (See FIG. 9).

In order to determine the amount of B. oleracea DNA still present in theBC₃ plants generated from RQ 5000/06, a DNA marker analysis with 72 SSRmarkers covering the whole C-genome of B. napus was performed. Theanalysis (example 14) revealed that the BC₃ plants had very highhomology to E. sativa, and that all of the B. oleracea chromosomes hadbeen eliminated in the back-crossing process.

EXAMPLE 2 CMS E. sativa Generated by Protoplast Fusion

A. Seed Sterilization and Germination

Seeds of B. oleracea or B. napus with CMS are dipped for approximately10 seconds in 70% alcohol and sterilized in a 1.5% sodium hypochloritesolution for two times for 10 minutes at 20° C. The sterilised seeds arethen extensively rinsed with sterile distilled water. The seeds are thenplaced on the MS nutrient medium (see table 1) with 3% sucrose andwithout hormones. To obtain green sterile plants, the seeds are grown inglass jars in the light (8000 lux), for a 16 hour photoperiod withtemperatures of 25° C. day and 20° C. night. Sterile shoots aresubcultured under the same conditions in plastic containers. To obtainwhite tissue for protoplast isolation, e.g. hypocotyls, the seeds aregrown in petri dishes in the dark at 25° C.

B. Isolation of Protoplasts

Leaves of four-week old shoots of plant material according to example 3are cut into small pieces and incubated in an enzyme solution (table 4A) for 16 hours at 25° C. on a gyratory shaker at 40 rpm. The suspensionis filtered through a nylon mesh (40 μm) and washed with CPW 16Ssolution (table 4D) by centrifugation at 817 rpm for 5 minutes. Thisresults in flotation of the intact protoplasts. The protoplasts arecollected and rinsed twice with W5 solution (table 4B) by centrifugationat 708 rpm for 5 minutes. The protoplasts are diluted to a density of1×10⁵ protoplasts per ml W5 solution before being used for fusionexperiments.

TABLE 4 A. ENZYME SOLUTION (1 L) 90 g mannitol 0.0272 g KH₂ PO₄ 0.1 gKNO₃ 0.246 g MgSO₄•7H₂O 0.0008 g KI 0.00025 g CuSO₄•5H₂O 1.4 g CaCl₂2H₂O 1.1 g MES 6 g Cellulose R10 1 g pH 5.8 Macarozyme B. WASHINGSOLUTION (W5) (1 L) 18.4 g CaCl₂•2H₂O 4.91 g NaCl 0.372 g KCl 0.901 gglucose pH = 5.8 C. WASHING SOLUTION (W5′) (1 L) 18.4 g CaCl₂•2H₂O 4.91g NaCl 0.372 g KCl 0.901 g glucose 9.76 g MES pH = 5.8 D. CPW 16 S (1L), 160 g sucrose 0.0272 g KH₂ PO₄ 0.1 g KNO₃ 1.45 g CaCl₂•2H₂O 0.246 gMgSO₄•2H₂O 0.0008 g KI 0.025 mg CuSO₄•5H₂O, pH = 5.5-5.8 E. PEG fusionsolution (PFS), 18.8% PEG (MW 4000) 0.06 M CaCl₂•2H₂O 0.1 M mannitol,0.025 M glucine, 10% (v/v) DMSO F. SPA SOLUTION (1 L) 20 g SeaPlaqueagarose, 100 g SucroseC. Preparation of Fluorescent-Stained Protoplasts

Six- to eight-day old hypocotyls of the plant material preparedaccording to example 2A are isolated according to the process of example2B, except that 3 μg/ml of fluoresceinediacetate is added to the enzymesolution used during the enzyme treatment step. Stained protoplastssuitable for hand selection and for determination of fusion frequencyare thus obtained.

D. Irradiation of Protoplasts

Freshly isolated protoplasts prepared according to example 2B are platedin a 6 cm petri dish in 2 to 3 ml W5 solution (table 4B). Theprotoplasts are irradiated using an X-ray source (Baltograph CE100), ata dose of 3500 Gy during 20 minutes. After irradiation, the inactivatedprotoplasts are washed with W5 solution by centrifugation at 708 rpm for5 minutes. The protoplasts are diluted to a density of 1×10⁵ protoplastsper ml W5 solution before being used for fusion experiments.

E. Fusion Procedure

Protoplasts according to example 2B, C and D, are mixed 1:3 in a finalconcentration of 1×10⁵ protoplasts per ml W5 solution. Three droplets of100 μl of the suspended protoplasts are placed in an uncoated 6 cm petridish and the protoplasts are allowed to settle for 5 to 10 minutes.Three hundred μl of PFS solution is added in the center of the threedroplets to induce agglutination for 15 minutes. Thereafter, 300 μl ofW5 solution is added to the mixture, and again after 10 minutes, andagain after a further 5 minutes. The W5 medium is replaced by two timesconcentrated 8P solution (table 1) and the protoplasts are cultivatedfor one to three days at 25° C. in the dark.

F. Selection and Growth of Fusion Products

The entire fusion mixture according to example 2E is cultured for one tothree days in the dark at 25° C. The cells are collected bycentrifugation at 548 rpm for 5 minutes and diluted in two timesconcentrated 8P medium to a density of 1×10⁵ protoplasts per ml. Anequal volume of 37° C. SPA medium (table 4F) is added and the cells areplated in 5 droplets of 100 μl in a coated petri dish C). Also 5droplets of 100 μl with feeder cells (cell suspension that improveregeneration) are added. After two weeks, the droplets with feeder cellsare removed (By using an pipette) and 2 ml MAC medium (table 1) perpetri dish is added. After two weeks the droplets are dispersed on solidBr medium (table 1). After two to three weeks the individual coloniesare transferred to a petri dish with solid K3 medium (table 1). Themicrocalli are cultured in low light intensity (2500 lux) at 25° C. witha photoperiod of 18 hours.

Fused cells, which can be recognized visually, for example by thepresence of double fluorescence, are picked up with a micromanipulator.The hybrid cells are cultured in 100 μl agarose droplets (1% SeaPlaque)at a density of 2000 to 50,000 protoplasts per milliliter. The dropletsare placed in a liquid nurse culture system (Costar-Transwell col) withfeeder cells and incubated at 25° C. in the dark. The droplets aredispersed on solid Br medium (table 1) after two weeks. Small calli aretransferred to solid K3 medium (table 1) and incubated at 25° C. in lowlight intensity (2500 lux) with a photoperiod of 16 hours.

G. Plant Regeneration

The calli according to examples 10 and 11, having developed to a size of2 to 5 mm in diameter, are transferred to fresh K3 medium (table 1) atnormal light intensity (8000 lux) at 25° C. with a photoperiod of 16hours. Small shoots are transferred to B5 medium (table 1) with 1%sucrose without hormones and rooted on the same medium.

EXAMPLE 3 CMS Identification by DNA Markers

A DNA marker was used to detect the presence of “Ogura” CMS in plantmaterial. Detection of the marker was based on a Polymerase ChainReaction (PCR) procedure employing the primer combination oligo37 andoligo38 (table 5). This primer combination yields a 512 bp PCR fragmentwhen the “Ogura” CMS-marker “orf138” is present.

TABLE 5 Oligo 37  5′GCA TCA CTC TCC CTG TCG TTA TCG3′ (upper primer):Oligo 38  5′ATT ATT TTC TCG GTC CAT TTT CCA3′ (lower primer): Cob gene TCT TCT CTC GGG GTC ATC CT (upper primer): Cob gene CCC CCT TCA ACA TCT CTC AT (lower primer):

The PCR was performed on total DNA extracted from 16 samples consistingof cauliflower with and without “Ogura” CMS, normal male fertilecultivars of E. sativa and F1 and BC₁ crosses between CMS-B. oleraceaand E. sativa. As an internal positive control a 630 bp marker (“cobgene”) was used.

All analysed plant samples were first ground in liquid nitrogen. DNA wasthen extracted from 250 mg plant material from each sample by means of aCTAB based DNA extraction protocol (Eurofins, Chen D. H. & Ronald, P.C., 1999). Average DNA yield was 5 ng/μL. Standard PCR conditions wereused with an annealing temperature of 53° C. The products of the PCRperformed on the extracted DNA from each plant sample employing the twoprimer pairs (oligos 37/38 and cob gene primers) were analyzed on a 1.5%agarose gel stained with ethidium bromide, as shown in FIG. 5.

A DNA fragment of 512 bp was positively detected in the threecauliflower lines known to carry “Ogura” CMS (FIG. 5A, lanes 1, 4b, 5),while the 512 bp fragment was absent from 2 non-“Ogura” cauliflowerlines (FIG. 5, lanes 2, 3b) and the 3 E. sativa lines (FIG. 5A, lanes 6,7, 8b). The 8 DNA extracts from samples of different generation crossesbetween Cauliflower F1 hybrid cv. “Cheddar” and E. sativa (FIG. 5A,lanes 9-16) were all positive for the 512 bp fragment, indicating thepresence of the orf138-gene in these interspecific hybrids.

The 5 BC₂ plants of line RQ 5000/06 also tested positive for the 512 bpfragment, indicating the presence of the orf138 CMS marker in line RQ5000/06 (see FIG. 5B).

EXAMPLE 4 Homology Studies by SSR Markers

Genomic DNA was extracted from five BC₃ plants generated from Line RQ5000/06, and analysed with a set of 72 markers.

The markers used met the following criteria:

-   -   all markers map on the C-genome of the B. napus genetic map    -   8 markers cover each of the 9 linkage groups (chromosomes) of B.        oleracea (the CMS donor, with C-genome).    -   the markers cover the C-genome of B. napus as evenly as possible

The DNA fragment analysis was performed as a multi loading assayanalyzing two or three markers, each labelled with a different ABI-dye,simultaneously. The test was designed to reveal the degree of homologybetween the BC₃ plants and the RP (E. sativa cv. “Myway”).

47 of the markers were not amplified from DNA of E. sativa recurrentparent (RP), whereas only 2 of the markers were not amplified from DNAof the B. oleracea CMS donor plant. All of the markers were amplifiedfrom DNA of the B. napus containing the C-genome, which was included asa positive control. In total 4 of the markers were non informative(including the 2 that did not amplify in B. oleracea) because theyshowed an exact match between the B. oleracea CMS donor and the E.sativa RP. Therefore, they were discarded. A positive match is countedas an exact match between the amplification products of two samples. Ifa marker was not amplified in either the RP (E. sativa) or the BC₃plants, this was also scored as a positive match.

TABLE 6 Percentage of exact positive matches between markeramplification products of the BC₃ plants derived from line RQ 5000/06,the B. oleracea CMS donor and the E. sativa RP. % match to % match to B.oleracea* E. sativa* B. napus 3 0 B. oleracea 100 0 Plant 20 (BC₃) fromRQ 5000/06 0 75 Plant 21 (BC₃) from RQ 5000/06 0 75 Plant 22 (BC₃) fromRQ 5000/06 0 75 Plant 23 (BC₃) from RQ 5000/06 0 75 Plant 24 (BC₃) fromRQ 5000/06 0 82 *The match percentage indicates the homology between theBC₃ plants, the CMS donor and the RP.

The match percentage indicates the homology between the BC₃ plants, theCMS donor and the RP. From table 6, it is seen that the BC₃ plants showa very high homology to E. sativa. The reason that the match is not 100%between the BC plants and the RP is that the RP is an open-pollinatedvariety and therefore it is not completely homozygotic. If all partialmatches, i.e. a match in one of multiple amplified DNA fragments from agiven marker, are included, the homology to E. sativa is between 85-90%in the BC₃ plants. None of the BC₃ plants show any homology to the CMSdonor. It is therefore concluded that all the chromosomes of B. oleraceahave been eliminated during the BC process.

REFERENCES

-   Cardi, T. & E. D. Earle, 1997. Production of new CMS Brassica    oleracea by transfer of “Anand” cytoplasm from B. rapa through    protoplast fusion, Theoretical and Applied Genetics, 94, 204-212-   Cargill, Inc., PO Box 9300, Minneapolis, Minn., 55440-9300, USA,    www.cargill.com-   Chen, D. H. & Ronald, P. C.; 1999. A rapid DNA minipreparation    method suitable for AFLP and other PCR applications. Plant Molecular    Reporter, 1999, p. 53-57.-   Glimelius, 1984, Physiologia Plantarum 61:38-   Hauptmann et al., 1983 “Carrot×Tobacco Somatic Cell Hybrids Selected    by Amino Acid Analog Resistance Complementation”, 6th International    Protoplast Symposium, Basel, Aug. 12-16,-   INRA, Agrocampus Rennes, BP 35327, F-35653 Le Rheu, France INRA,    NCBI, acc. Z12626-   Koop et al. 1989. Electroporation and Electrofusion in Cell Biology,    edited: Neuman et al. pgs 355-365-   L. Dæhnfeldt A/S, Faaborgvej 248B, 5250 Odense SV., Denmark-   Seminis Vegetable Seeds, Oxnard, Calif., USA, www.seminis.com-   Sigareva, M. A. & E. D. Earle, 1997, Direct transfer of a    cold-tolerant “Ogura” male sterile cytoplasm into cabbage (Brassica    oleracea ssp. Capitata) via protoplast fusion, Theoretical and    Applied Genetics, 94, 213-220-   Sundberg et al., 1986, Plant Science, 43:155-   Sundberg, E. & Glimelius, K., 1991. Effects of parental ploidy level    and genetic divergence on chromosome elimination and chloroplast    segregation in somatic hybrids within Brassicaceae. Theoretical and    Applied Genetics, vol. 83, no. 1, p. 81-82.-   Syngenta Seeds B. V., Westeinde 62, P.O. Box 2, 1600 AA Enkhuizen,    Netherlands. www.syngenta.com-   TraitGenetics GmbH, Am Schwabeplan 1b, D-06466 Gatersieben, Germany-   Tsunoda S., Hinata K. & Gómez-Campo C., 1980, Brassica crops and    wild allies, Japan Scientific Societies Press, Tokyo-   Yadav, R. C., Yadav, N. R. & P. K. Sareen, 2002. Interspecific    hybridization in different Brassica species: pollen tube studies.    National Journal of Plant Improvement, vol. 4, p. 42-47.

1. A method of producing a male sterile Eruca sativa plant, wherein saidplant comprises a male sterile cytoplasm conferring male sterility uponsaid plant, comprising the steps of: a) crossing a cytoplasmic malesterile Brassica oleracea [or a cytoplasmic male sterile Brassica napus]plant comprising Ogura male sterile cytoplasm with an Eruca sativaplant, b) rescuing one or more embryos resulting from the cross of stepa), c) regenerating a plant from one or more embryos of step b) andselecting one or more plant having Eruca sativa phenotype, d) doublingthe chromosome number of the plant of step c), and e) back-crossing oneor more plant resulting from step d) with an Eruca sativa plant andselecting for one or more plant with Eruca sativa phenotype.
 2. Themethod according to claim 1, further comprising back-crossing one ormore plant resulting from step e) with an Eruca sativa plant andselecting for one or more plant with Eruca sativa phenotype.
 3. Themethod according to claim 1, wherein said selecting further includestesting for an orf138 DNA marker and wherein said selected plantcomprises said marker.
 4. The method according to claim 2 furthercomprising crossing said one or more selected plant to an Eruca sativainbred to produce one or more hybrid plant.